Tumor heterogeneity poses a distinct challenge to effective treatment of cancer patients. Next generating sequencing over the past decade has revealed how the ways in which tumor heterogeneity leads to worse outcomes for patients, by enabling tumors to evolve and develop resistance to targeted therapies. This project aims to investigate the origins of tumor heterogeneity, making use of a mouse model system that is designed to enable the identification of distinct tumor subpopulations and genetic analysis of these populations. This project will combine the disciplines of tumor progression and metastasis, tumor microenvironment studies, and cancer genetics to provide novel insights into the nature of subpopulations within a tumor and evolution of these subpopulations during the course of a tumor's progression from a benign lesion to aggressively metastatic disease. It will be completed primarily by a graduate student with significant experience in mouse carcinogenesis models and next generation sequencing over the course of two years, under the supervision of a faculty sponsor with decades of experience with skin carcinogenesis and cancer genetics. Objectives: This proposal will (1) study the nature of subpopulations of cells that are found in early tumors, determining whether they are separately arising tumor cells or come from the tumor's microenvironment; (2) determine the behavior and contributions of tumor subpopulations during the course of progression from benign to metastatic disease. This work will provide a foundation for the development of cancer therapies better suited to combatting deadly heterogeneous tumors. Two specific aims are proposed: Aim 1: Test hypothesis that tumors which contain multiple contributing populations are recruiting normal tissue from the microenvironment. This aim will study subpopulations that are present within tumors at the earliest stages of tumor development, with the goal of understanding their origins and the long-term contributions they make to the tumor. Aim 2: Determine clonality and growth dynamics of early- and late-stage tumor progression, including the timing of metastatic dissemination. This aim will study tumor subpopulations that develop after the tumor is initiated. Subpopulations will be labeled at 8, 16, or 24 weeks after tumor initiation and their behavior studied as the tumor progresses to malignancy and metastasizes. Labeling subpopulations at different time points will further allow interrogation as to when tumors metastasize, a question that has significant impact of patient treatment strategies.